The adeno-associated viruses (AAV) are members of the family Parvoviridae and the genera Dependoviruses. Serotypes 1 through 4 were originally identified as contaminates of adenovirus preparations (Carter and Laughlin (1984) in, The Parvoviruses p. 67-152 New York, N.Y.) whereas type 5 was isolated from a patient wart that was HPV positive. To date, twelve molecular clones have been generated representing the serotypes of human/primate AAV (Bantel-Schaal et al. (1999) J. Virol. 73: 939; Chiorini at al. (1997) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Mori et al. (2004) Virol. 330:375; Muramatsu et al. (1996) Virol. 221:208; Ruffing et al. (1994) J. Gen. Virol. 75:3385; Rutledge et al. (1998) J. Virol. 72:309; Schmidt et al. (2008) J. Virol. 82:8911; Srivastava et al. (1983) J. Virol. 45:555; Xiao et al. (1999) J. Virol. 73:3994). These clones have provided valuable reagents for studying the molecular biology of serotype specific infection. Transduction of these viruses naturally results in latent infections, with completion of the life cycle generally requiring helper functions not associated with AAV viral gene products. As a result, all of these serotypes are classified as non-pathogenic and are believed to share a safety profile similar to the more extensively studied AAV type 2 (Carter and Laughlin (1984) in, The Parvoviruses p. 67-152 New York, N.Y.).
General understanding of the mechanisms required for function at origins of replication has grown immensely since the first prokaryotic origins were characterized. While the DNA-protein interactions necessary for replication in prokaryotes, lower eukaryotes, and bacteriophages are generally well understood, mechanisms employed in the majority of higher eukaryotes and vertebrate viruses, such as AAV, are still being determined. The inverted terminal repeats (ITRs) of AAV and other Parvoviruses act as the origin of replication. These elements flank the short, single stranded genome and typically possess a T-shaped secondary structure. The replication strategies of the genus Dependovirus, including those of AAV, have been well characterized. The viral non-structural or Replication proteins (Rep) are the only factors required to interact with the ITR in order to catalyze replication (Im and Muzyczka (1990) Cell 61:447). The majority of AAV serotypes possess highly conserved origins of replication with interchangeable DNA-protein interactions. However, the Rep proteins of several serotypes interact exclusively with their cognate ITR. Discovering the mechanisms which drive Rep-ITR specificity promises to advance our understanding of DNA-protein interactions at viral origins of replication. These findings also promise to shed light on how eukaryotic and prokaryotic proteins achieve selectivity to DNA substrates.
The AAV rep gene encodes four multifunctional proteins (Hermonat et al. (1984) J. Virol. 51:329; Tratschin et al. (1984) J. Virol. 51:611; Mendelson et al. (1986) J. Virol. 60:823; Trempe et al. (1987) Virol. 161:18) that are expressed from two promoters at map units 5 (p5) and 19 (p19). The larger Rep proteins transcribed from the p5 promoter (Rep78 and Rep68), are essentially identical except for unique carboxy termini generated from unspliced (Rep78) and spliced (Rep68) transcripts, respectively (Srivastava et al, (1983) J. Virol. 45:555). The two smaller Rep proteins, Rep52 and Rep40, are transcribed from the p19 promoter and represent amino terminal truncations of Rep78 and Rep68, respectively.
Several biochemical activities of Rep78 and Rep68 have been characterized as involved in AAV replication. These include specific binding to the AAV ITR (Ashktorab et al. (1989) J. Virol. 63:3034; Im et al. (1989) J. Virol. 63:3095; Snyder et al. (1993) J. Virol. 67:6096) and site-specific endonuclease cleavage at the terminal resolution site (trs) (Im et al. (1990) J. Virol. 63:447; Im et al. (1992) J. Virol. 66:1119; Snyder et al., (1990) Cell 60:105; Snyder et al. (1990) J. Virol. 64:6204). Rep78/68 also possess ATP dependent DNA-DNA helicase (Im et al., (1990) J. Virol. 63:447; Im et al. (1992) J. Virol. 66:1119) and DNA-RNA helicase as well as ATPase activities (Wonderling et al. (1995) J. Virol. 69:3542). In addition to these activities involved in replication, Rep78/68 also regulate transcription from the viral promoters (Beaton et al. (1989) J. Virol. 63:4450; Labow et al. (1986) J. Virol. 60:251; Tratschin et al. (1986) Mol. Cell. Biol. 6:2884; Kyostio et al. (1994) J. Virol. 68:2947; Pereira et al. (1997) J. Virol. 71:1079), and have been shown to mediate viral targeted integration (Xiao, W., (1996), “Characterization of cis and trans elements essential for the targeted integration of recombinant adeno-associated virus plasmid vectors”, Ph.D. Dissertation, University of North Carolina-Chapel Hill; Balague et al. (1997) J. Virol. 71:3299; LaMartina et al. (1998) J. Virol. 72:7653; Pieroni et al. (1998) Virol. 249:249).
Like Rep proteins, the AAV ITRs are involved in nearly every aspect of the viral life-cycle. The secondary structure of the ITR is necessary to prime synthesis of the second strand to allow transcription of the viral genes (Hauswirth and Berns (1977) J. Virol. 78:488). The full length Rep proteins contain a unique N-terminal DNA binding region which specifically recognizes the ITR at the 16 nt Rep-binding element (RBE) and at the tip of one of the hairpin stems known as the RBE′ (FIG. 1A) (Ryan et al. (1996) J. Virol. 70:1542; Brister and Muzyczka (2000) J. Virol. 74:7762). Rep molecules multimerize on the ITR allowing the C-terminus of Rep, acting as an ATP-dependent SF3 helicase, to unwind the ITR and form a putative internal hairpin (Im and Muzyczka (1990) Cell 61:447; Hermonat and Batchu (1997) FEBS Lett. 20:180). This hairpin, (here, termed ‘nicking stem’) contains the terminal resolution site (trs) where Rep nicks the ITR in a site-specific manner (Brister and Muzyczka (1999) J. Virol. 73:9325). This DNA cleavage is important for replication of the closed ITR and to initiate subsequent rounds of genomic replication. Replicated genomes can undergo replication again or be encapsidated in the presence of the smaller Rep proteins (King et al. (2001) EMBO J. 20:3282).
The ITR sequences of twelve human/primate AAV serotypes have been published. These sequences typically display 80% or greater nucleotide conservation and segregate into two groups (Hewitt et al. (2009) J. Virol. 83:3919). The AAV2 Rep proteins (Rep2) are able to function on the ITR of every known AAV serotype except those of AAV5 (ITR5; Hewitt et al. (2009) J. Virol. 83:3919; Grimm et al. (2006) J. Virol. 80:426). Consistently, the AAV5 Rep proteins (Rep5) are unable to catalyze replication of the ITR of AAV2 (ITR2). Replicative specificity between these serotypes does not exist at the level of binding, as Rep2 and Rep5 can bind interchangeably to ITR2 or ITR5 (Chiorini et al. (1999) J. Virol. 73:4293). Instead, specificity is created by the inability of Rep to cleave the ITR of the opposite serotype. This occurs despite high conservation between the ITR2 and ITR5 sequence, secondary structure, and location of elements required for Rep interaction (RBE, RBE′, trs, nicking stem).
All current AAV vectors in clinical trials utilize ITR2s. However, using ITR2s for therapeutic purposes creates a safety risk due to the ubiquity of AAV2 in the human population as well as other AAVs whose Rep proteins can replicate ITR2s. In this manner, rAAV vectors have the potential to be “mobilized” out of the target tissue into different tissues of the body or into other individuals in the population (Hewitt et al. (2009) J. Virol. 83:3919).
The present invention provides a solution to vector mobilization through the creation of a novel Rep-ITR interaction. A vector utilizing this novel interaction cannot be mobilized by one or more of the wild-type AAV serotypes which infect humans, nor the non-human serotypes which can potentially infect human hosts.